Evidence for SN2(P) mechanism in the phosphorylation of alkaline phosphatase by substrates

Abstract

Michaelis–Menten parameters have been measured for a series of alkyl-, aryl-, and arylamido-phosphates as substrates of alkaline phosphatase. Kinetic parameters for p-bromophenylamido-phosphate were measured over a pH-range and the results, including inhibition by inorganic phosphate indicated the amides to be normal substrates of the enzyme. Little variation in the parameter k₀ was observed for all substrates; the parameter k₀/K_m which is a measure of the rate constant for phosphorylation of the free enzyme by free substrate showed a high selectivity to steric factor in the alkyl phosphate series. The amido-phosphates were more than ten-fold less reactive than the aryl phosphates in the k₀/K_m parameter and were calculated to be 60,000-fold too small for the S_N1 (P) mechanism. Both these results together with arguments taken from the literature point to an S_N2(P) mechanism for phosphorylation involving direct nucleophilic attack at phosphorus. The substituted arylamido-phosphates show a decreasing reactivity with electron-withdrawing substituent indicating acid catalysis. The zinc(II) necessary for phosphatase action is suggested to promote an S_N2(P) mechanism at phosphorus by complexing with the substrate and neutralising the negative charge on the oxygen thus activating the phosphorus to nucleophilic attack.